Construction and Transposon Mutagenesis in Escherichia Coli of a Full-length Infectious Clone of Pseudorabies Virus, an Alphaherpesvirus

Overview

Journal J Virol

Publisher American Society for Microbiology

Date 1999 Jul 10

PMID 10400733

Citations 148

Authors

G A Smith

L W Enquist

Affiliations

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Abstract

A full-length clone of the 142-kb pseudorabies virus (PRV) genome was constructed as a stable F plasmid in Escherichia coli. The clone, pBecker1, was colinear with PRV-Becker genomic DNA, lacking detectable rearrangements, deletions, or inversions. The transfection of pBecker1 into susceptible eukaryotic cells resulted in productive viral infection. Virus isolated following transfection was indistinguishable from wild-type virus in a rodent model of infection and spread to retinorecipient regions of the brain following inoculation in the vitreous body of the eye. Mutagenesis of pBecker1 in E. coli with a mini-Tn5-derived transposon enabled the rapid isolation of insertion mutants, identification of essential viral genes, and simplified construction of viral revertants. The serial passage of a viral insertion mutant demonstrated the transposon insertion to be stable. However, the F-plasmid insertion present in the viral gG locus was found to undergo a spontaneous deletion following transfection into eukaryotic cells. The implications of F-plasmid insertion into the viral genome with regard to phenotype and genomic stability are discussed.

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References

Card J, Rinaman L, Schwaber J, Miselis R, Whealy M, Robbins A . Neurotropic properties of pseudorabies virus: uptake and transneuronal passage in the rat central nervous system. J Neurosci. 1990; 10(6):1974-94. PMC: 6570305. View

OConnor M, Peifer M, Bender W . Construction of large DNA segments in Escherichia coli. Science. 1989; 244(4910):1307-12. DOI: 10.1126/science.2660262. View

Peeters B, de Wind N, Hooisma M, Wagenaar F, Gielkens A, Moormann R . Pseudorabies virus envelope glycoproteins gp50 and gII are essential for virus penetration, but only gII is involved in membrane fusion. J Virol. 1992; 66(2):894-905. PMC: 240790. DOI: 10.1128/JVI.66.2.894-905.1992. View

Shizuya H, Birren B, Kim U, Mancino V, Slepak T, Tachiiri Y . Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc Natl Acad Sci U S A. 1992; 89(18):8794-7. PMC: 50007. DOI: 10.1073/pnas.89.18.8794. View

McNabb D, Courtney R . Characterization of the large tegument protein (ICP1/2) of herpes simplex virus type 1. Virology. 1992; 190(1):221-32. DOI: 10.1016/0042-6822(92)91208-c. View

McNabb D, Courtney R . Analysis of the UL36 open reading frame encoding the large tegument protein (ICP1/2) of herpes simplex virus type 1. J Virol. 1992; 66(12):7581-4. PMC: 240474. DOI: 10.1128/JVI.66.12.7581-7584.1992. View

Heffner S, Kovacs F, Klupp B, Mettenleiter T . Glycoprotein gp50-negative pseudorabies virus: a novel approach toward a nonspreading live herpesvirus vaccine. J Virol. 1993; 67(3):1529-37. PMC: 237523. DOI: 10.1128/JVI.67.3.1529-1537.1993. View

Cohen J, Seidel K . Generation of varicella-zoster virus (VZV) and viral mutants from cosmid DNAs: VZV thymidylate synthetase is not essential for replication in vitro. Proc Natl Acad Sci U S A. 1993; 90(15):7376-80. PMC: 47140. DOI: 10.1073/pnas.90.15.7376. View

Cunningham C, Davison A . A cosmid-based system for constructing mutants of herpes simplex virus type 1. Virology. 1993; 197(1):116-24. DOI: 10.1006/viro.1993.1572. View

10.

Tomkinson B, Robertson E, Yalamanchili R, Longnecker R, Kieff E . Epstein-Barr virus recombinants from overlapping cosmid fragments. J Virol. 1993; 67(12):7298-306. PMC: 238193. DOI: 10.1128/JVI.67.12.7298-7306.1993. View

11.

Enquist L, Dubin J, Whealy M, Card J . Complementation analysis of pseudorabies virus gE and gI mutants in retinal ganglion cell neurotropism. J Virol. 1994; 68(8):5275-9. PMC: 236473. DOI: 10.1128/JVI.68.8.5275-5279.1994. View

12.

de Lorenzo V, Timmis K . Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons. Methods Enzymol. 1994; 235:386-405. DOI: 10.1016/0076-6879(94)35157-0. View

13.

Babic N, Mettenleiter T, Ugolini G, Flamand A, Coulon P . Propagation of pseudorabies virus in the nervous system of the mouse after intranasal inoculation. Virology. 1994; 204(2):616-25. DOI: 10.1006/viro.1994.1576. View

14.

Kempkes B, Pich D, Zeidler R, Sugden B, Hammerschmidt W . Immortalization of human B lymphocytes by a plasmid containing 71 kilobase pairs of Epstein-Barr virus DNA. J Virol. 1995; 69(1):231-8. PMC: 188568. DOI: 10.1128/JVI.69.1.231-238.1995. View

15.

Alexeyev M, Shokolenko I, Croughan T . New mini-Tn5 derivatives for insertion mutagenesis and genetic engineering in gram-negative bacteria. Can J Microbiol. 1995; 41(11):1053-5. DOI: 10.1139/m95-147. View

16.

Kemble G, Duke G, Winter R, Spaete R . Defined large-scale alterations of the human cytomegalovirus genome constructed by cotransfection of overlapping cosmids. J Virol. 1996; 70(3):2044-8. PMC: 190037. DOI: 10.1128/JVI.70.3.2044-2048.1996. View

17.

Knapp A, Enquist L . Pseudorabies virus recombinants expressing functional virulence determinants gE and gI from bovine herpesvirus 1.1. J Virol. 1997; 71(4):2731-9. PMC: 191395. DOI: 10.1128/JVI.71.4.2731-2739.1997. View

18.

Card J, Dubin J, Whealy M, Enquist L . Influence of infectious dose upon productive replication and transynaptic passage of pseudorabies virus in rat central nervous system. J Neurovirol. 1995; 1(5-6):349-58. DOI: 10.3109/13550289509111024. View

19.

Messerle M, Crnkovic I, Hammerschmidt W, Ziegler H, Koszinowski U . Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci U S A. 1998; 94(26):14759-63. PMC: 25110. DOI: 10.1073/pnas.94.26.14759. View

20.

Tirabassi R, Enquist L . Role of envelope protein gE endocytosis in the pseudorabies virus life cycle. J Virol. 1998; 72(6):4571-9. PMC: 109969. DOI: 10.1128/JVI.72.6.4571-4579.1998. View